Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Genomic DNA: Genomic DNA from mOSNs or MOE was isolated using the DNeasy Kit (Qiagen) using the standard protocol for cells and tissues, respectively. RNA: Total RNA was isolated from a given cell population or tissue using TRIzol (Invitrogen), then processed differently for FACS-isolated mOSNs or whole MOE. For mOSN, the aqueous phase of the TRIzol extraction was passed through a Zymo RNA Clean and Concentrator 5 column, DNase-treated on column using 1 unit of TURBO DNase for 15 minutes at 37°C, and eluted twice using 10 μL water. For whiole MOE, total RNA isolation was continued using the standard isopropanol precipitation, resuspension in water, and incubation for 20 minutes at 37°C with 1U TURBO DNase (Ambion). Native chromatin: Standard: Genomic DNA was sonicated to 200-1000 bp with a mean size of ~400 bp and end repaired (NEBNext End Repair). Adenosine was added to 3' ends (Klenow, 3' to 5' exo-, NEB), and 10:1 Illumina adaptor:samples were ligated onto the 200-500 ng samples. Ribominus/Scriptseq: 200 ng of total RNA was rRNA-depleted using two rounds of RiboMinus (Invitrogen) followed by library construction using ScriptSeq v2 (Epicentre) according to manufacturer protocol and amplification for 15 cycles using Phusion (NEB) and provided primers. Nugen encore complete: 100 ng of total RNA was processed using the Encore Complete RNA-seq system (Nugen) and amplified for 15 cycles using Phusion (NEB) and provided primers. Nugen Ovation Ultralow Input: Post ChIP DNA was sonicated to a mean size of 300 bp and processed using the Ovation Ultralow Input Kit (Nugen) RNA-seq: Total RNA was isolated as described in extract protocol section and prepared for sequencing as described in library construction protocol section. ChIP-seq: Nuclei were isolated from the MOEs of 3-week old Dnmt3a wildtype and knockout mice and digested with micrococcal nuclease (MNase, Sigma) to yield a population of mono- to tetra- nucleosomes that was, subsequently, used in chromatin immunoprecipitation assays using anti-H3K27ac (Millipore, cma309), anti-H3K4me1 (Abcam, ab8895), or anti-H3K27me3 (Millipore, 07-449) antibodies. Immunoprecipitated DNA was isolated by phenol-chloroform extraction. For library preparation, ChIP DNA was sonicated for 120-180 seconds on a Covaris S220 and prepared for sequencing using the Ovation Ultralow Library Kit (Nugen). MeDIP-seq: Genomic DNA was isolated, prepared for paired-end Illumina sequencing as indicated in library construction section. 20-100 ng of adapted genomic DNA was diluted in MB (10 mM phosphate buffer pH 7.0, 140 mM NaCl, 0.05% Triton X-100), denatured at 95°C for 10 minutes, brought to 300 µL MB, and precipitated with 0.5 μg anti-5mC (Active Motif, 39649) or 5hmC (Active Motif, 39791) antibodies overnight at 4°C. Separately, 10 µL per IP of Protein A and G Dynabeads each (Invitrogen) were blocked with 2 mg BSA and 2 mg yeast tRNA in 1 mL MB overnight at 4°C. Beads and IPs were combined the next day and rotated for 3 hours at 4°C. Samples were washed 4 times in MB, eluted twice in 100 µL elution buffer (0.1 M NaHCO3, 1% SDS, 1 mM EDTA) by shaking at 37°C for 15 minutes, phenol:chloroform:isoamyl extracted, ethanol precipitated, and resuspended in 50 µL TE. Samples were then amplified with Illumina TruSeq PCR primers for 15 cycles using Phusion DNA polymerase (NEB). DNase-hypersensitivity: Nuclei from the olfactory epithelium were isolated as described for ChIP-seq and resuspended in nuclease digestion buffer (0.32 M sucrose, 50 mM Tris-HCl pH 7.5, 4 mM MgCl2, 1 mM CaCl2, 0.1 mM PMSF). 3 million nuclei were brought to 250 μL nuclease digestion buffer, pre-warmed for one minute at 37°C, and incubated with 20 U DNase I (Ambion) for 2 or 5 minutes. Reactions were stopped with the addition of 28 μL 0.5 M EDTA and placement on ice. Reactions were then incubated with 200 μg proteinase K (Ambion) 55°C overnight, extracted using phenol:chloroform:isoamyl (Invitrogen), and ethanol precipitated. Samples were resuspended in TE, incubated with 10 μg RNase A (Roche) at 37°C for 30 minutes, extracted using phenol:chloroform:isoamyl (Invitrogen), and ethanol precipitated. Samples were resuspended in TE, and one half of each reaction was run on a 1% agarose/1x TAE gel. Regions 100-500 bp were excised and purified using Qiagen Gel Extraction Kit. Illumina sequencing libraries were then prepared as described in library construction section and amplified for 9 cycles using Phusion DNA polymerase (NEB) and Illumina TruSeq primers.